The long-term objective of this proposal is to understand in molecular terms the mechanism by which Agrobacterium tumefaciens interacts with its host resulting in Crown Gall tumor formation in a wide variety of plants. This proposal follows the same general approach that we have used in previous years to gain insight into these mechanisms, namely, to analyze mutants altered in tumor formation. However, the experiments described are much more focused because we now know the sequence of the Agrobacterium genome and we have access to microarray chip technology. With this new information and technology we can carry out directed mutant studies and types of analyses that were not possible previously. The information revealed by the sequence of the genome has raised questions but also the means to answer these questions which we could only speculate on previously. Thus, we now know that far more genes are activated in Agrobacterium by interaction with host plants than was recognized previously. Using microarray chip technology we will identify and characterize global regulatory circuits that are directly activated or suppressed by plant signal molecules. Using microarray technology, we will expand on previous observations by determining the alterations in the global expression of genes in Agrobacterium grown with plant cells at a temperature optimum for the growth of Agrobacterium but inhibitory for the transfer of T-DNA (28). We will also continue our studies on the mechanism by which T-DNA is transferred from Agrobacterium into host cells, focusing on the role of VirJ in this process. Finally, the genome sequence of Agrobacterium has revealed many genes that serve pathogenicity functions in related bacteria. We will mutate these genes to determine if they are also required for pathogenicity of Agrobacterium. The studies proposed in this application should open up new vistas and provide an increased understanding of the many levels of interaction of Agrobacterium with its variety of eukaryotic hosts.